U.S. patent application number 09/923563 was filed with the patent office on 2002-08-22 for method for improved production of cyanophycin and secondary products thereof.
Invention is credited to Berg, Holger, Ebert, Jan, Lockau, Wolfgang, Piotukh, Kirill, Volkmer-Engert, Rudolf, Ziegler, Karl.
Application Number | 20020115141 09/923563 |
Document ID | / |
Family ID | 7651791 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115141 |
Kind Code |
A1 |
Ziegler, Karl ; et
al. |
August 22, 2002 |
Method for improved production of cyanophycin and secondary
products thereof
Abstract
The present invention relates to a thermostable cyanophycin
synthetase produced from Synechococcus elongatus and to a method
for improved production of cyanophycin and/or secondary products
thereof.
Inventors: |
Ziegler, Karl; (Berlin,
DE) ; Lockau, Wolfgang; (Berlin, DE) ; Ebert,
Jan; (Berlin, DE) ; Piotukh, Kirill; (Moskau,
RU) ; Berg, Holger; (Berlin, DE) ;
Volkmer-Engert, Rudolf; (Berlin, DE) |
Correspondence
Address: |
BAYER CORPORATION
PATENT DEPARTMENT
100 BAYER ROAD
PITTSBURGH
PA
15205
US
|
Family ID: |
7651791 |
Appl. No.: |
09/923563 |
Filed: |
August 7, 2001 |
Current U.S.
Class: |
435/69.1 ;
435/219; 435/252.3; 435/254.2; 435/410 |
Current CPC
Class: |
C12N 9/93 20130101; C12P
21/02 20130101; C07K 14/00 20130101 |
Class at
Publication: |
435/69.1 ;
435/219; 435/252.3; 435/254.2; 435/410 |
International
Class: |
C12P 021/02; C12N
009/50; C12N 001/18; C12N 005/04; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 2000 |
DE |
10038775.6 |
Jul 27, 2001 |
EP |
PCT/EP01/08690 |
Claims
We claim:
1. A cyanophycin synthetase comprising an amino acid sequence
according to SEQ ID No: 01, encoded by a nucleotide sequence
according to SEQ ID No: 02, an allele, homologue or derivative of
said nucleotide sequence or a nucleotide sequence hybridizing
therewith, and originating from Synechococcus elongatus, wherein
said cyanophycin synthetase has a temperature optimum of at least
35.degree. C.
2. A vector comprising at least one nucleotide sequence coding for
a cyanophycin synthetase according to claim 1 or 19, which is
specific for cyanophycin production.
3. A transformed unicellular or multicellular organism comprising a
cyanophycin synthetase according to claim 19 and/or a vector
according to claim 2.
4. A transformed unicellular or multicellular organism according to
claim 3, wherein said organism is selected from the group
consisting of a microorganism, a fungus, a lower or higher plant,
tissue or at least one cell therefrom.
5. A method for providing a cyanophycin synthetase according to
claim 1 or 19, comprising the steps of operatively linking said
nucleotide sequence coding for said cyanophycin synthetase to
regulatory structures and/or is cloning said nucleotide sequence
into a vector suitable for heterologous expression, transferring
said nucleotide sequence into a heterologous host system and
expressing and isolating and/or purifying and/or concentrating said
cyanophycin synthetase from said host system.
6. A method for producing cyanophycin and the secondary products to
be produced therefrom, comprising the step of employing a
cyanophycin synthetase according to claim 1 or 19 and/or a vector
according to claim 2 and/or a transformed unicellular or
multicellular organism according to any of claims 3 and 4 to
produce said cyanophycin.
7. A method for preparing a transformed unicellular or
multicellular organism according to either of claims 3 and 4
comprising the step of employing a vector according to claim 2 to
produce said transformed organism.
8. A method for producing a compound selected from the group
consisting of a cyanophycin synthetase according to claim 1 or 19
and cyanophycin comprising the steps of providing the transformed
unicellular or multicellular organism according to any of claims 3
to 4, whereupon said organism produces said cyanophycin synthetase
and/or said cyanophycin.
9. A method for producing cyanophycin comprising the steps of
providing said cyanophycin synthetase according to claim 1 or 19,
and employing said cyanophycin synthetase to produce said
cyanophycin.
10. Isoenzymes and modified forms of cyanophycin synthetase wherein
said isoenzymes and said modified forms of cyanophycin synthetase
are obtained by modifying cyanophycin synthetasefrom Synechococcus
elongatus and wherein said iso enzymes and modified forms of
cyanophycin synthetase have a temperature optimum in the range of
35.degree. C. to 55.degree. C.
11. The isoenzymes and the modified forms of cyanophycin synthetase
according to claim 10, wherein said isoenzymes and said modified
forms of cyanophycin synthetase are obtained by amino acid
exchange.
12. The isoenzymes and the modified forms of cyanophycin synthetase
according to claim 11, wherein the amino acid exchange is carried
out by modifying a nucleotide sequence of an underlying gene of
said Synechococcus elongatus.
13. An artificial DNA sequence, comprising an artificial DNA
sequence that is insertable into or appendable to a gene wherein
said artificial DNA sequence encodes for and expresses the
cyanophycin synthetase according to claim 1 or 19.
14. A probe for the identification and/or isolation of one or more
genes coding for proteins involved in cyanophycin biosynthesis,
said probe comprising a label suitable for detecting cyanophycin
synthetase and modifications thereof according to claims 1 and
10.
15. A heterologous host system comprising a nucleic acid sequence
or a part thereof coding for a member of the group consisting of
cyanophycin synthetase, an isoenzyme and modified forms thereof
according to claims 1, 19 or 10.
16. A method for the synthesis of polyaspartic acid or arginine
comprising the steps of providing said cyanophycin synthetase
according to claim 1 or 19 and employing said cyanophycin
synthetase to produce said polyaspartic acid or said arginine.
17. Cyanophycin synthetase comprising a DNA sequence selected from
the group consisting of a natural DNA sequence and an artificial
DNA sequence, said DNA sequence being located between the 5' or
upstream and/or 3' or downstream position of the cyanophycin
synthetase according to claim 1, wherein said DNA sequences
influence transcription, RNA stability of RNA processing, and
translation.
18. The synthetase of claim 1 wherein said synthetase has a
temperature optimum in the range of about 35.degree. C. to about
55.degree. C.
19. The synthetase of claim 1 wherein said synthetase has a
temperature optimum in the range of 35.degree. C. to 55.degree.
C.
20. The synthetase of claim 1 wherein said synthetase has a
temperature optimum in the range of 35.degree. C. to 50.degree.
C.
21. A polypeptide having cyanophycin synthetase functionality
comprising a cyanophycin synthetase having a temperature optimum of
at least 35.degree. C. and wherein at least one amino acid sequence
of said polypeptide has been altered such that said polypeptide is
rendered insensitive to the regulating action of regulatory
compounds that would otherwise regulate the activity of said
polypeptide with respect to said polypeptide's cyanophycin
synthetase functionality.
22. The polypeptide of claim 21 wherein said cyanophycin synthetase
is a cyanophycin synthetase according to claims 1 or 19.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to a thermostable cyanophycin
synthetase, to transformed organisms containing such an enzyme and
to a method for improved production of cyanophycin and/or secondary
products thereof, for example polyaspartic acid or arginine.
BACKGROUND OF THE INVENTION
[0002] Multi-L-arginyl-poly-L-aspartate (cyanophycin) is a branched
polypeptide which contains aspartic acid and arginine in the ratio
of 1:1. The chemical structure corresponds to a
poly-.alpha.-aspartate backbone with arginine side radicals which
are linked via peptide bonds to virtually all .beta.-carboxyl
groups of the backbone. DE-A 198 25 509 describes the
identification, cloning and heterologous expression of the gene for
cyanophycin synthetase from Synechocystis PCC 6803. The enzyme
activity is determined here by means of a radioactive assay in
which L-[U-.sup.14C]-arginine is incorporated into cyanophycin from
Aphanocapsa PCC 6308, introduced as primer. The enzyme reaction
itself takes place at 28.degree. C. here.
[0003] DE-A 197 09 024 discloses the extraction and purification of
cyanophycin from Aphanocapsa PCC 6308, the synthesis being carried
out at 20.degree. C.
[0004] DE-A 198 13 692 merely discloses isolation of the
cyanophycin synthetase gene from Synechocystis PCC 6803 or Anabaena
variabilis ATCC 29 413. Technical aspects of cyanophycin
production, however, are not described here.
[0005] FEMS Microbiology Letters 181 (1999) 229-236 discloses the
production of cyanophycin using Synechococcus sp. MA 19.
[0006] A disadvantage of large-scale cyanophycin production
according to the known methods is that, for optimal product yield,
a relatively narrow temperature range, normally below 35.degree.
C., should not be exceeded.
[0007] This represents a considerable restriction in the degrees of
freedom for large-scale production within the process control for
the production of cyanophycin, since higher temperatures from the
outset prevent contamination by foreign cultures.
[0008] Therefore production of cyanophycin also at substantially
higher temperatures than previously described, in combination with
higher flexibility in process control and considerably improved
product yields is desirable, in order to isolate therefrom the
secondary products such as polyaspartic acid or arginine on a large
scale.
[0009] This object is achieved by the present invention.
SUMMARY OF THE INVENTION
[0010] The present invention relates to a cyanophycin synthetase
which is distinguished by having a temperature optimum in the range
of >35.degree. C. and an amino acid sequence according to SEQ ID
No: 01, encoded by an isolated nucleotide sequence according to SEQ
ID No: 02, an allele, homologue or derivative of this nucleotide
sequence or a nucleotide sequence hybridizing therewith.
[0011] In a preferred variant of the present invention, the
cyanophycin synthetase of the invention has a temperature optimum
in the range from 35.degree. C. bis 55.degree. C., preferably in
the range from 35.degree. C. bis 50.degree. C.
[0012] The cyanophycin synthetase is further distinguished by the
fact that it originates from Synechococcus elongatus. The
cyanophycin synthetase of the invention represents a thermostable
enzyme.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a representation of the chemical structure of the
synthetic peptide primers used for synthesis of cyanophycin by
means of cyanophycin synthetase.
[0014] FIG. 2 is a representation of the results of an SDS
polyacrylamide gel electrophoresis (SDS-PAGE) for in vitro
synthesis of cyanophycin-like material by means of purified
cyanophycin synthetase.
[0015] FIG. 3 is a representation of the results of an SDS-PAGE for
chain elongation of a primer by means of cyanophycin synthetase at
the C-terminal end of the peptide primer.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0016] The present invention relates to a cyanophycin synthetase
which is distinguished by having a temperature optimum in the range
of >35.degree. C. and an amino acid sequence according to SEQ ID
No: 01, encoded by an isolated nucleotide sequence according to SEQ
ID No: 02, an allele, homologue or derivative of this nucleotide
sequence or a nucleotide sequence hybridizing therewith.
[0017] In a preferred variant of the present invention, the
cyanophycin synthetase of the invention has a temperature optimum
in the range from 35.degree. C. bis 55.degree. C., preferably in
the range from 35.degree. C. bis 50.degree. C.
[0018] The cyanophycin synthetase is further distinguished by the
fact that it originates from Synechococcus elongatus. The
cyanophycin synthetase of the invention represents a thermostable
enzyme.
[0019] The present invention also relates to isoenzymes of the
cyanophycin synthetase of the invention. These isoenzymes mean
enzymes having identical or comparable substrate specificity and
action specificity, but having a different primary structure. In
addition, the present invention also includes modified forms of
cyanophycin synthetase. According to the invention, these mean
enzymes in which alterations are present in the sequence, for
example at the N and/or C termini of the polypeptide or in the
region of conserved amino acids, which alterations, however, do not
impair the function of the enzymes. These modifications may be
carried out by exchanging one or more amino acids according to
known methods.
[0020] A particular embodiment of the present invention includes
variants of the inventive cyanophycin synthetase, whose substrate
specificity, for example, was altered, for example with regard to
the production of polyaspartic acid, by the amino acid exchange,
compared with the particular starting protein. The same is true for
the stability of the enzymes of the invention in cells; for
example, the enzymes have increased or reduced sensitivity towards
degradation by proteases.
[0021] The present invention further relates to polypeptides with
cyanophycin synthetase function, whose amino acid sequence has been
altered such that they are insensitive to regulatory compounds, for
example to the metabolic endproducts regulating their activity
(feedback insensitive).
[0022] An isolated nucleotide sequence or an isolated nucleic acid
fragment means, according to the invention, an RNA or DNA polymer
which may be single- or double-stranded and may optionally contain
natural, chemically synthesized, modified or artificial
nucleotides. The term "DNA polymer" here also includes genomic DNA,
cDNA or mixtures thereof.
[0023] According to the invention, alleles mean functionally
equivalent nucleotide sequences, i.e. nucleotide sequences with
essentially identical action. Functionally equivalent sequences are
those sequences which, despite deviating nucleotide sequences, for
example due to the degeneracy of the genetic code, still retain the
desired functions. Functional equivalents thus include naturally
occurring variants of the sequences described herein and also to
artificial nucleotide sequences obtained, for example, by chemical
synthesis and, where appropriate, adjusted to the codon usage of
the host organism. Moreover, functionally equivalent sequences
include those having a modified nucleotide sequence which confers
on the enzyme insensitivity or resistance to inhibitors, for
example.
[0024] A functional equivalent means in particular also natural or
artificial mutations of an originally isolated sequence which
continue to show the desired function. Mutations include
substitutions, additions, deletions, exchanges or insertions of one
or more nucleotide residues. Also included here are "sense
mutations" which can lead at the protein level to the exchange of
conserved amino acids, for example, but not to any fundamental
change in the protein activity and thus are functionally neutral.
This also includes modifications of the nucleotide sequence which,
at the protein level, concern the N or C terminus of a protein but
with no substantial restriction of protein function. These
modifications may even have a stabilizing influence on the protein
structure.
[0025] The present invention further also includes those nucleotide
sequences which are obtained by modification of the nucleotide
sequence, resulting in corresponding derivatives. The aim of such a
modification may be, for example, the further narrowing down of the
coding sequence contained therein or else, for example, the
introduction of further recognition sites for restriction enzymes.
Functional equivalents are also those variants whose function,
compared with the starting gene or gene fragment, is reduced or
enhanced.
[0026] In addition, the present invention relates to artificial DNA
sequences, as long as they provide the desired properties, as
described above, and can be inserted into or appended to the gene
of the cyanophycin synthetase of the invention. It is possible, for
example, to determine such artificial DNA sequences by translating
back from proteins generated by means of computer-assisted programs
(molecular modelling) or by in vitro selection. Coding DNA
sequences which have been obtained by translation back from a
polypeptide sequence according to the codon usage specific for the
host organism are particularly suitable. It is possible for a
skilled worker familiar with molecular genetic methods readily to
determine the specific codon usage by computer analyses of other,
already known genes of the organism to be transformed.
[0027] According to the invention, homologous sequences mean those
which are complementary to the nucleotide sequences of the
invention and/or hybridize with these sequences. The term
"hybridizing sequences" includes, according to the invention,
substantially similar nucleotide sequences from the group
comprising DNA or RNA, which specifically interact with (bind to)
the abovementioned nucleotide sequences under known stringent
conditions. This also includes short nucleotide sequences of, for
example, from 10 to 30, preferably from 12 to 15 nucleotides in
length. According to the invention, "nucleotide primers" or probes
are inter alia also included here.
[0028] According to the invention, the sequence regions preceding
(5' or upstream) and/or following (3' or downstream) the coding
regions (structural genes) are also included. In particular,
sequence regions with regulatory function are included here. They
can influence transcription, RNA stability or RNA processing and
also translation. Examples of regulatory sequences are inter alia
promoters, enhancers, operators, terminators or translation
enhancers.
[0029] Operative linkage means the sequential arrangement of, for
example, promoter, coding sequence, terminator and, where
appropriate, further regulatory elements, such that each of the
regulatory elements can fulfil its predetermined function when the
coding sequence is expressed. These regulatory nucleotide sequences
may be of natural origin or can be obtained by chemical synthesis.
A suitable promoter is in principle any promoter which is able to
control gene expression in the appropriate host organism. According
to the invention, the said promoter may also be a chemically
inducible promoter which makes it possible to control at a
particular time expression of the genes subject to it in the host
cell. By way of example, mention may be made here of a promoter
inducible by IPTG (isopropyl .beta.-thiogalactopyranoside).
[0030] A gene construct is prepared by fusion of a suitable
promoter with the nucleotide sequence of the invention, according
to common recombination and cloning techniques known from the
literature. The DNA fragments can be linked to one another by
attaching adapters or linkers to the fragments.
[0031] Moreover, the present invention relates to a vector
comprising at least one nucleotide sequence of the type described
above coding for a cyanophycin synthetase specific for producing
cyanophycin, regulatory nucleotide sequences operatively linked to
the said nucleotide sequence and also additional nucleotide
sequences for selection of transformed host cells, for replication
within the host cell or for integration into the appropriate host
cell genome. The vector of the invention may further comprise a
gene construct of the abovementioned type.
[0032] Suitable vectors are those which are replicated in
micro-organisms such as, for example, bacteria, fungi and/or
plants. Examples of known vectors are pBluescript (Stratagene,
11099 North Torney Pines Rd., La Jolla, Calif. 92 037, USA) or pET
(Novagen, 601 Science Drive, Madison, WJ 53 711, USA). This list,
however, is non-limiting for the present invention.
[0033] Utilizing the nucleic acid sequences of the invention, it is
possible to synthesize and use appropriate probes or else
nucleotide primers for the purpose of amplifying and isolating
analogous genes from other unicellular or multicellular organisms,
preferably bacteria, fungi, algae or plants, for example with the
aid of the polymerase chain reaction ("PCR") technique.
[0034] The present invention thus also relates to a probe for
identifying and/or isolating genes coding for proteins involved in
cyanophycin biosynthesis, preferably further thermostable
cyanophycin synthetases; this probe is prepared starting from the
inventive nucleic acid sequences of the type described above and
contains a label suitable for detection. The probe may be a section
of the sequence of the invention, for example from a conserved
region, which is, for example, from 10 to 30 or, preferably, 12 to
15 nucleotides in length and which can hybridize specifically with
homologous nucleotide sequences under stringent conditions.
Suitable labels are known from the literature in large numbers.
[0035] The present invention further relates to the transfer of the
inventive nucleic acid sequence or a part thereof, coding for a
cyanophycin synthetase, an allele, homologue or derivative thereof,
or of a nucleotide sequence hybridizing with these sequences into a
heterologous host system. This also includes the transfer of a gene
construct or vector of the invention into a heterologous host
system.
[0036] According to the invention, a heterologous host system means
a unicellular or multicellular organism. Examples of these are
micro-organisms, fungi, lower or higher plants, tissue or cells
thereof. According to the invention, preference is given to
bacteria, particularly preferably of the genus of enterobacteria
and, in particular, of the species Escherichia coli. Furthermore,
useful plants such as potatoes or tobacco are particularly
preferred.
[0037] The inventive nucleotide sequence coding for an inventive
thermostable cyanophycin synthetase is transferred into one of the
abovementioned host systems according to known methods. Examples of
methods for DNA transfer into suitable host systems, which may be
mentioned, are transformation, electroporation, conjugation and
agrobacteria-mediated DNA transfer or particle bombardment. This
list serves only the purpose of illustrating the present invention
and is non-limiting.
[0038] A transformed unicellular or multicellular organism
resulting from a successful nucleic acid transfer thus differs from
the corresponding untransformed organism by containing and being
able to express additional nucleic acids of the inventive type.
[0039] The invention thus also relates to a transformed unicellular
or multicellular organism comprising a cyanophycin synthetase of
the invention and/or a vector comprising a cyanophycin synthetase
of the type described above.
[0040] The present invention further relates to a method for
providing an inventive cyanophycin synthetase of the type described
above, in which method the nucleotide sequence coding for the
enzyme is isolated from a thermophilic unicellular or multicellular
organism, is, where appropriate, operatively linked to regulatory
structures and/or cloned into a vector suitable for heterologous
expression, is, where appropriate, transferred into a heterologous
host system, is expressed there and is finally isolated from this
host system and, where appropriate, purified and/or
concentrated.
[0041] Direct isolation of an amount of cyanophycin synthetase
which is sufficient for cyanophycin synthesis, from a thermophilic
organism, without prior concentration in a heterologous system, is
also conceivable. Furthermore, it is then possible to use the
inventive cyanophycin synthetase enzyme, for example in an in vitro
system for synthesizing cyanophycin and/or secondary products
thereof.
[0042] The present invention also relates to a method for producing
cyanophycin and/or secondary products thereof, in which a
cyanophycin synthetase and/or a vector and/or a transformed
unicellular or multicellular organism of the type described above
are used. However, the present invention includes not only the
production of cyanophycin and/or secondary products thereof in a
living host system but also the in-vitro synthesis of cyanophycin
with the aid of an isolated cyanophycin synthetase of the type
described above.
[0043] The inventive method for producing cyanophycin is
distinguished by carrying out the enzyme-catalyzed synthesis in a
temperature range from 35.degree. C. to 55.degree. C., preferably
in a range from 35.degree. C. to 50.degree. C.
[0044] The method of the invention is advantageously distinguished
by the fact that, owing to the wide temperature range, the process
is less error-prone, in particular above 28.degree. C., allows
greater variability in process control and thus provides improved
product yield. Thus, the inventive production of cyanophycin and/or
secondary products thereof is substantially more reproducible and
economical than the hitherto known methods.
[0045] At the molecular level, the cyanophycin synthetase of the
invention catalyses an ATP-dependent chain elongation.
Surprisingly, the enzyme has two active (catalytic) centres. The
cyanophycin synthetase of the invention stepwise and alternately
(sequentially) incorporates one aspartic acid molecule and
subsequently one arginine molecule into a cyanophycin precursor
(peptide primer). Without a primer, the enzyme-catalysed chain
elongation cannot be started. Studies thereon are depicted in FIG.
2.
[0046] Referring now to FIG. 2 there is illustrated a
representation of the results of an SDS polyacrylamide gel
electrophoresis (SDS-PAGE) for in vitro synthesis of
cyanophycin-like material by means of purified cyanophycin
synthetase. The reaction mixture contains inter alia about 10 .mu.M
Primer (.beta.-Asp-Arg).sub.3. After incubation for 24 hours at
room temperature, aliquots of the reaction mixture are analysed by
means of SDS-PAGE and proteins are visualized according to standard
methods. The lanes illustrate the following: Lane 1: complete
reaction mixture; lane 2: reaction mixture without aspartic acid;
lane 3: reaction mixture without arginine; lane 4: reaction mixture
without ATP; lane 5: reaction mixture without primer
(.beta.-Asp-Arg).sub.3; lane 6: reaction mixture with
heat-inactivated enzyme (5 min, 100.degree. C.). The protein band
above the 97.4 kDa standard represents cyanophycin synthetase. The
diffuse bands below 29 kDa in lanes 1, 2 and 3 represent
cyanophycin-like material.
[0047] The chemical structure of various primers used in the
synthesis of cyanophycin is depicted in FIG. 1. This clearly
indicates that incorporation takes place exclusively at the
C-terminal end of the precursor and only if both amino acids, i.e.
aspartic acid and arginine or another basic amino acid, are present
together. A summary of these studies is depicted in FIG. 3 and
Table 1.
[0048] Referring now to FIG. 3 there is illustrated a
representation of the results of an SDS-PAGE for chain elongation
of a primer by means of cyanophycin synthetase at the C-terminal
end of the peptide primer. Various primers (FIG. 1) are added to
the reaction mixture. After incubation of the reaction mixtures for
24 hours at room temperature, aliquots of the reaction mixture are
analysed by means of SDS-PAGE and proteins are visualized according
to standard methods. The lanes illustrate the following: lane 1:
mixture without primer; lane 2: mixture with about 8 .mu.M
unprotected primer (.beta.-Asp-Arg).sub.3; lane 3: mixture with
about 8 .mu.M N-terminally protected primer
(.epsilon.-Ahx.sub.2-(.beta.-Asp-Arg).sub.3); lane 4: mixture with
about 8 .mu.M C-terminally protected primer
((.beta.-Asp-Arg).sub.3-.epsilon.-A- hx.sub.2); lane 5: as lane 4
but with about 160 .mu.M primer. The diffuse bands below 29 kDa in
lanes 1, 2 and 3 represent cyanophycin-like material. Table 1 below
illustrates the cyanophycin synthetase-catalysed incorporation of
L-aspartic acid (Asp) and L-arginine (Arg) into synthetic peptide
primers.
1TABLE 1 No. Primer Substrate(s) Product C-terminally blocked
primer: (1) (.beta.-Asp-Arg).sub.3-.epsilon.-Ahx.sub.2 Asp + Arg
none (2) (.beta.-Asp-Arg).sub.3-.epsilon.-Ahx.sub.2 Asp or Arg none
N-terminally blocked primer: (3) .epsilon.-Ahx.sub.2-(.beta.-Asp-A-
rg).sub.3 Asp + Arg Cyanophycin (4)
.epsilon.-Ahx.sub.2-(.beta.-Asp- -Arg).sub.3 Asp
.epsilon.-Ahx.sub.2-(.beta.-Asp-Arg).sub.3-Asp (5)
.epsilon.-Ahx.sub.2-(.beta.-Asp-Arg).sub.3 Arg none Unblocked
primers: (6) (.beta.-Asp-Arg).sub.3 Asp + Arg Cyanophycin (7)
(.beta.-Asp-Arg).sub.3 Asp (.beta.-Asp-Arg).sub.3-Asp.sup.a) (8)
(.beta.-Asp-Arg).sub.3 Arg none (9) (.beta.-Asp-Arg).sub.3
.beta.-Asp-Arg none (10) (.beta.-Asp-Arg).sub.3-Asp Asp + Arg
Cyanophycin (11) (.beta.-Asp-Arg).sub.3-Asp Asp none (12)
(.beta.-Asp-Arg).sub.3-Asp Arg (.beta.-Asp-Arg).sub.4 .sup.a)In
principle, the reaction product could also be
Asp-(.beta.-Asp-Arg).sub.3. However, this possibility is excluded
due to the results using the N-terminally or C-terminally blocked
primers (reactions 2 and 4 of this table).
[0049] The present invention further relates to the use of a vector
comprising an inventive cyanophycin synthetase of the
abovementioned type for preparing a transformed unicellular or
multicellular organism as described above. The present invention
likewise includes the use of such a transformed unicellular or
multicellular organism for producing an inventive cyanophycin
synthetase and/or for producing cyanophycin and/or secondary
products thereof. Moreover it is also possible to make use of a
cyanophycin synthetase isolated according to the invention for
in-vitro production of cyanophycin and/or secondary products
thereof. In addition, the present invention relates to the use of
cyanophycin and/or secondary products thereof for producing food
supplements and/or compositions in the fields of agriculture and/or
crop protection. Further fields of application for cyanophycin
and/or secondary products thereof can be found in the paper,
textile, pigment, paint, ceramics, building material or detergent
industry and also in the fields of water and wastewater
treatment.
[0050] The present invention is characterized in more detail by the
following examples which are, however, not limiting for the
invention:
[0051] General Genetic Methods:
[0052] DNA isolation, plaque hybridization, polymerase chain
reaction (PCR), construction of a genomic DNA gene library and the
procedures for protein analysis by means of SDS polyacrylamide gel
electrophoresis (SDS-PAGE) including protein purification, as well
as culturing of microorganisms such as, for example, Escherichia
coli are carried out according to standard methods described in
Sambrook, J. et al. (1998, Molecular Cloning: A Laboratory Manual;
2.sup.nd Edition, Cold Spring Harbor Laboratory Press, N.Y.) or
according to information by the manufacturers. Blue-green algae
such as, for example, Synechococcus elongatus were cultured
according to descriptions in Yamaoka, T., et al. (1978, Plant Cell
Physiol., 19: 943-954).
[0053] Peptide Primer Synthesis:
[0054] The branched peptide primers (.beta.-Asp-Arg).sub.3,
(.beta.-Asp-Arg).sub.3-Asp, .epsilon.-Ahx.sub.2-(b-Asp-Arg).sub.3
and (.beta.-Asp-Arg).sub.3-.epsilon.-Ahx.sub.2 (see FIG. 1;
Ahx=.epsilon.-aminohexane acid) were synthesized on a solid phase
following Fmoc/tBu chemistry via
O-(benzotriazol-1-yl)-N,N,N',N'-tetramet- hyluronium
tetrafluoroborate activation using the building block
Fmoc-Asp-[Arg(Pmc)-OtBu]-OH. The building block was prepared in
solution by the following reaction sequence: (i) acylation of
H-Arg(Pmc)-OtBu (Bachem Biochemicals) with Fmoc-Asp-All (All=allyl
ester) (Novabiochem) using
dicyclohexylcarbodiimide/N-hydroxybenzotriazole (Novabiochem) as
activators and then (ii) opening of the allyl ester with the aid of
N-methylaniline and tetrakis(triphenylphosphine)palladium(0) as
catalyst. The synthesis is started on a resin loaded with
Fmoc-Arg(Pmc)-TentaGel-S-- PHB (Rapp Polymere). The peptide primers
are linked by the following reaction: (i) coupling of Fmoc-Asp-OtBu
and subsequently (ii) attaching twice the building block
Fmoc-Asp-[Arg-(Pmc)-OtBu]-OH. Furthermore, the N-terminally blocked
peptide primer .epsilon.-Ahx.sub.2-(b-Asp-Arg).sub.3 was prepared
by attaching Fmoc-.epsilon.-aminohexane acid (Novabiochem) twice to
the resin-bound peptide primer described above. The finished
peptides were deprotected by treatment with 94% trifluoroacetic
acid, 1% phenol, 2% water, 3% triisobutylsilane and removed from
the resin, the peptides and the N-terminally blocked peptide
primers being obtained as free acids. The C-terminally blocked
peptide primer (.beta.-Asp-Arg).sub.3-.epsilon.-Ahx.sub.2 was
prepared on a TentaGel-SRAM resin (Rapp-Polymere) according to the
following procedure: (i) two times coupling of
Fmoc-.epsilon.-aminohexane acid and subsequently three times
coupling of the building block Fmoc-L-Asp-[L-Arg(Pmc)-OtBu). The
finished primer was removed as described above and gave the peptide
as carboxamide.
[0055] The peptide primer (.beta.-Asp-Arg).sub.3-Asp was
synthesized on a TentaGel-S-PHB resin (Rapp Polymere) loaded with
Fmoc-Asp(OtBu) by attaching the appropriate building block three
times. As described above the peptide was likewise removed from the
resin and deprotected. The peptide was obtained as free acid here.
All peptide primers were purified on a C-18 column (Vydac 201SP54)
and analysed with the aid of RP HPLC and MALDI MS.
[0056] The dipeptide .beta.-Asp-Arg was likewise prepared on a
TentaGel-S-PHB phase which had been loaded with Fmoc-Arg(Pmc)
before. After the Fmoc protection group had been removed with 20%
strength piperidine-DMF solution, the resin was treated with 4 eq
(equivalents) of Boc-Asp-OtBu (Bachem Chemicals), 4 eq of
O-(benzotriazol-1-yl)-N,N,N',N'-- tetramethyluronium
tetrafluoroborate and 8 eq of diisopropyl-ethylamine in DMF. The
peptide was removed from the resin with trifluoroacetic acid
containing 1% phenol, 2% water and 3% triisobutylsilane, then
precipitated with cold t-butylmethyl and finally purified on a C-18
column (Vydac 201SP54) and analysed by RP HPLC and MALDI MS.
[0057] Reaction Mixtures and Product Analysis:
[0058] The reaction mixtures for product analysis by means of mass
spectrometry contain in a volume of 125 .mu.l the following
components: 100 mM NH.sub.4HCO.sub.3 (pH 8.0), 4 mM ATP disodium
salt, 20 mM MgCl.sub.2, 8 mM KCl, 2 mM DTT, 0.2 mM L-aspartic acid,
0.2 mM L-arginine, .gtoreq.10 .mu.M synthetic primers and 3 .mu.g
of cyanophycin synthetase.
[0059] For product analysis by means of SDS-PAGE, 125 .mu.l of
reaction mixture contain the following: 50 mM Tris-HCl (pH 8.0), 4
mM ATP disodium salt, 20 mM MgCl.sub.2, 20 mM KCl, 1 mM DTT, 0.8 mM
L-aspartic acid, 0.4 mM L-arginine, .gtoreq.10 .mu.M synthetic
primers and 3 .mu.g of cyanophycin synthetase.
[0060] The samples are incubated at room temperature for 10-14
hours and subsequently either mixed with sample buffer (SDS-PAGE)
or frozen (mass spectrometry). The products are analysed by means
of mass spectrometry (MALDI MS) according to the information in the
user manual of the manufacturer (PerSeptive Biosystems).
[0061] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients,
reaction conditions, etc. used in the specification and claims are
to be under stood as modified in all instance by the term
"about."
[0062] Although the invention has been described in detail in the
foregoing for the purpose of illustration, it is to be understood
that such detail is solely for that purpose and that variations can
be made therein by those skilled in the art without departing from
the spirit and scope of the invention except as it may be limited
by the claims.
Sequence CWU 1
1
2 1 896 PRT Synechococcus elongatus 1 Met Lys Ile Leu Lys Leu Gln
Thr Leu Arg Gly Pro Asn Tyr Trp Ser 1 5 10 15 Ile Arg Arg His Lys
Leu Ile Val Met Arg Leu Asp Leu Glu Glu Val 20 25 30 Ala Asn Thr
Pro Ser Asn Gln Ile Ser Gly Phe Val Asp Gly Leu Val 35 40 45 Arg
Val Leu Pro Ser Leu Tyr Asn His Phe Cys Ser Leu Gly His Glu 50 55
60 Gly Gly Phe Leu Thr Arg Leu Arg Glu Gly Thr Tyr Leu Gly His Val
65 70 75 80 Val Glu His Val Ala Leu Glu Leu Gln Glu Leu Ala Gly Met
Pro Val 85 90 95 Gly Phe Gly Arg Thr Arg Glu Thr Ser Thr Pro Gly
Val Tyr Gln Val 100 105 110 Val Tyr Glu Tyr Gln Val Glu Glu Ala Gly
Arg Tyr Ala Gly Arg Ala 115 120 125 Ala Val Arg Leu Cys Gln Ser Ile
Ile Asp Thr Gly Thr Tyr Pro Gln 130 135 140 Gln Glu Leu Asp Gln Asp
Leu Ala Asp Leu Arg Glu Leu Lys Ala Lys 145 150 155 160 Ala Ser Leu
Gly Pro Ser Thr Glu Ala Ile Val Arg Glu Ala Glu Ala 165 170 175 Arg
Asn Ile Pro Trp Phe Glu Leu Ser Ser Arg Ser Ile Ile Gln Leu 180 185
190 Gly Tyr Gly Ala Arg Ser His Arg Met Gln Ala Thr Leu Ser Asp Arg
195 200 205 Ser Ser Ile Leu Ala Val Glu Leu Ala Ser Asp Lys Glu Gly
Ala Lys 210 215 220 Arg Leu Leu Gln Asp Ala Gly Ile Pro Val Pro Lys
Gly Thr Val Ile 225 230 235 240 Arg Tyr Ile Glu Asp Leu Pro Glu Ala
Ile Glu Glu Ile Gly Gly Tyr 245 250 255 Pro Ile Val Ile Lys Pro Leu
Asn Gly Asn His Gly Arg Gly Ile Thr 260 265 270 Ile Asp Ile Asn Ser
Leu Glu Ala Ala Glu Glu Ala Phe Glu Ile Ala 275 280 285 Ser Ser Ile
Ser Lys Ser Val Ile Val Glu Arg Tyr His Ala Gly Arg 290 295 300 Asp
Phe Arg Val Leu Val Val Asn Gly Lys Val Val Ala Val Ala Glu 305 310
315 320 Arg Val Pro Ala His Val Ile Gly Asp Gly His Ser Thr Ile Glu
Glu 325 330 335 Leu Ile Glu Lys Thr Asn Gln Asp Pro Gln Arg Gly Asp
Gly His Asp 340 345 350 Asn Ile Leu Thr Arg Ile Glu Val Asn His Asp
Thr Trp Thr Leu Leu 355 360 365 Glu Lys Gln Gly Tyr Thr Leu Asn Thr
Val Leu Gln Pro Gly Glu Ile 370 375 380 Cys Tyr Leu Arg Ala Thr Ala
Asn Leu Ser Thr Gly Gly Ile Ala Ile 385 390 395 400 Asp Arg Thr Asp
Glu Ile His Pro Glu Asn Val Trp Ile Cys Gln Arg 405 410 415 Ala Ala
Arg Ile Ile Gly Leu Asp Ile Ala Gly Ile Asp Val Val Ser 420 425 430
Pro Asp Ile Ser Gln Pro Leu Ser Lys Val Gly Gly Val Ile Val Glu 435
440 445 Val Asn Ala Ala Pro Gly Phe Arg Met His Thr Asn Pro Ser Gln
Gly 450 455 460 Ile Ala Arg Asn Val Ala Glu Pro Val Leu Asn Met Leu
Phe Pro Pro 465 470 475 480 Gly Thr Pro Cys Arg Ile Pro Ile Phe Ala
Ile Thr Gly Thr Asn Gly 485 490 495 Lys Thr Thr Thr Thr Arg Leu Ile
Ala His Ile Cys Lys Gln Thr Gly 500 505 510 Gln Thr Val Gly Tyr Thr
Thr Thr Asp Gly Ile Tyr Ile Gly Asp Tyr 515 520 525 Leu Val Glu Lys
Gly Asp Thr Thr Gly Pro Gln Ser Ala Gln Leu Ile 530 535 540 Leu Gln
Asp Pro Thr Val Glu Ile Ala Val Leu Glu Thr Ala Arg Gly 545 550 555
560 Gly Ile Leu Arg Ser Gly Leu Gly Phe Asp His Cys Asp Val Gly Val
565 570 575 Val Leu Asn Val Gln Ala Asp His Leu Gly Leu Gly Asp Ile
Asp Thr 580 585 590 Val Glu Gln Leu Ala Asp Leu Lys Ala Val Val Val
Glu Ser Ala Trp 595 600 605 Pro Asn Gly Tyr Ala Val Leu Asn Ala Asp
Asp Pro Leu Val Ala Ala 610 615 620 Met Ala Arg Gln Val Lys Ala Gln
Val Ala Tyr Phe Ser Met Asp Pro 625 630 635 640 His Asn Pro Ile Ile
Arg Gln His Ile Gln Gln Gly Gly Leu Ala Ala 645 650 655 Val Tyr Glu
Asn Gly Tyr Leu Ser Ile Leu Lys Gly Asp Trp Thr Leu 660 665 670 Arg
Ile Glu Gln Ala Glu Asn Val Pro Ile Thr Leu Gly Ala Arg Ala 675 680
685 Ser Phe Met Ile Ala Asn Ala Leu Ala Ala Ser Leu Ala Ala Phe Ala
690 695 700 Gln Gly Ile Ser Ile Glu His Ile Arg Ala Ala Leu Thr Thr
Phe Arg 705 710 715 720 Thr Ser Val Glu Gln Thr Pro Gly Arg Met Asn
Leu Phe Asp Leu Gly 725 730 735 Gln Phe Ser Val Leu Val Asp Tyr Ala
His Asn Pro Ala Gly Tyr Glu 740 745 750 Ala Ile Gly Glu Phe Val Gln
Lys Trp Pro Gly Gln Arg Ile Gly Val 755 760 765 Val Gly Gly Pro Gly
Asp Arg Arg Asp Gln Asp Leu Glu Gln Leu Gly 770 775 780 Glu Leu Ser
Ala Lys Ile Phe Asp Trp Ile Ile Ile Lys Glu Asp Asp 785 790 795 800
Asp Thr Arg Gly Arg Pro Arg Gly Asp Ala Ala Tyr Trp Ile Glu Arg 805
810 815 Gly Val His His His Ser Val Gln Arg Gln Tyr Asp Ile Ile His
Asp 820 825 830 Glu Val Ala Ala Ile Gln Phe Ala Leu Asp Arg Ala Pro
Lys Gly Ser 835 840 845 Leu Val Val Ile Phe Pro Ala Glu Val Ser Arg
Thr Ile Gln Leu Ile 850 855 860 Arg Gln His His Gln Arg Leu Gln Gly
Glu Thr Ile Asn Gly Phe His 865 870 875 880 Ser Glu Gly Arg Pro Thr
Ser Gly Asp Leu Asn Pro Ser Ile Phe His 885 890 895 2 2691 DNA
Synechococcus elongatus CDS (1)..(2691) cphA (cyanophycin
synthetase) 2 atg aag att ctc aaa tta caa acg ctg cgg ggt ccc aat
tac tgg agc 48 Met Lys Ile Leu Lys Leu Gln Thr Leu Arg Gly Pro Asn
Tyr Trp Ser 1 5 10 15 att cgg cgt cat aag ctg att gtc atg cgt tta
gat cta gaa gag gtg 96 Ile Arg Arg His Lys Leu Ile Val Met Arg Leu
Asp Leu Glu Glu Val 20 25 30 gcc aac acc ccc tcc aat cag att tct
ggg ttt gtg gat ggg ttg gtg 144 Ala Asn Thr Pro Ser Asn Gln Ile Ser
Gly Phe Val Asp Gly Leu Val 35 40 45 cgg gtt ttg ccg agt ctt tac
aat cat ttt tgt tct ctc gga cac gaa 192 Arg Val Leu Pro Ser Leu Tyr
Asn His Phe Cys Ser Leu Gly His Glu 50 55 60 ggg ggc ttt ctc acc
cgc ctc cga gaa ggt acg tat ctt ggt cat gtg 240 Gly Gly Phe Leu Thr
Arg Leu Arg Glu Gly Thr Tyr Leu Gly His Val 65 70 75 80 gtt gaa cat
gtt gcc ctc gag ctc caa gaa ctg gca ggg atg ccc gtt 288 Val Glu His
Val Ala Leu Glu Leu Gln Glu Leu Ala Gly Met Pro Val 85 90 95 ggc
ttt ggc cgc acg cgg gag acc tca acg ccg ggg gtg tat caa gtg 336 Gly
Phe Gly Arg Thr Arg Glu Thr Ser Thr Pro Gly Val Tyr Gln Val 100 105
110 gtc tat gaa tac caa gtg gaa gaa gcg ggc cgc tat gcc ggc cga gca
384 Val Tyr Glu Tyr Gln Val Glu Glu Ala Gly Arg Tyr Ala Gly Arg Ala
115 120 125 gca gtg cga ctg tgc caa agt att att gat acg ggt acc tat
ccc cag 432 Ala Val Arg Leu Cys Gln Ser Ile Ile Asp Thr Gly Thr Tyr
Pro Gln 130 135 140 caa gaa ctg gat cag gat ctc gcc gat ctc cgg gag
ttg aaa gca aaa 480 Gln Glu Leu Asp Gln Asp Leu Ala Asp Leu Arg Glu
Leu Lys Ala Lys 145 150 155 160 gcc tcc ctt ggc ccg agt acg gaa gcg
att gtc cgc gaa gcc gaa gcc 528 Ala Ser Leu Gly Pro Ser Thr Glu Ala
Ile Val Arg Glu Ala Glu Ala 165 170 175 cgc aac atc cct tgg ttt gag
ttg agc agt cgc tcg att att caa ttg 576 Arg Asn Ile Pro Trp Phe Glu
Leu Ser Ser Arg Ser Ile Ile Gln Leu 180 185 190 ggc tat ggc gcc cgc
agt cat cgg atg caa gcc aca ttg agc gat cgc 624 Gly Tyr Gly Ala Arg
Ser His Arg Met Gln Ala Thr Leu Ser Asp Arg 195 200 205 agt agc atc
ttg gca gtt gaa ctc gcc agt gac aaa gaa ggg gca aag 672 Ser Ser Ile
Leu Ala Val Glu Leu Ala Ser Asp Lys Glu Gly Ala Lys 210 215 220 cga
ctg ctt cag gat gcg gga att ccc gtg cct aag gga acc gtc atc 720 Arg
Leu Leu Gln Asp Ala Gly Ile Pro Val Pro Lys Gly Thr Val Ile 225 230
235 240 cgc tat att gaa gac ctc ccc gag gcc att gag gag atc ggt ggc
tat 768 Arg Tyr Ile Glu Asp Leu Pro Glu Ala Ile Glu Glu Ile Gly Gly
Tyr 245 250 255 ccc att gtc att aag ccc ctc aac ggc aac cac ggt cgc
ggg att acg 816 Pro Ile Val Ile Lys Pro Leu Asn Gly Asn His Gly Arg
Gly Ile Thr 260 265 270 att gac atc aac agc cta gaa gca gcc gaa gaa
gcc ttt gaa att gcc 864 Ile Asp Ile Asn Ser Leu Glu Ala Ala Glu Glu
Ala Phe Glu Ile Ala 275 280 285 agc agc atc tcc aaa tcc gtc att gtg
gaa cgc tat cat gcc ggt cgc 912 Ser Ser Ile Ser Lys Ser Val Ile Val
Glu Arg Tyr His Ala Gly Arg 290 295 300 gac ttc cgc gtt cta gtg gtc
aat ggc aaa gtg gtt gct gtt gct gaa 960 Asp Phe Arg Val Leu Val Val
Asn Gly Lys Val Val Ala Val Ala Glu 305 310 315 320 cgg gtg ccg gcc
cat gtg att ggc gat ggc cac tcc acc atc gaa gaa 1008 Arg Val Pro
Ala His Val Ile Gly Asp Gly His Ser Thr Ile Glu Glu 325 330 335 ctc
att gag aaa acg aac caa gac ccg caa cgg gga gac ggt cac gat 1056
Leu Ile Glu Lys Thr Asn Gln Asp Pro Gln Arg Gly Asp Gly His Asp 340
345 350 aat atc ctc acc cgc att gaa gtc aac cac gac act tgg aca ctc
ctg 1104 Asn Ile Leu Thr Arg Ile Glu Val Asn His Asp Thr Trp Thr
Leu Leu 355 360 365 gaa aaa cag ggc tat acc ctg aat acg gtc ttg caa
ccg ggg gaa att 1152 Glu Lys Gln Gly Tyr Thr Leu Asn Thr Val Leu
Gln Pro Gly Glu Ile 370 375 380 tgt tat cta cgg gcc acg gcg aac cta
agt act ggt ggc att gcc atc 1200 Cys Tyr Leu Arg Ala Thr Ala Asn
Leu Ser Thr Gly Gly Ile Ala Ile 385 390 395 400 gat cgc act gat gaa
att cac ccg gaa aat gtt tgg att tgc cag cgg 1248 Asp Arg Thr Asp
Glu Ile His Pro Glu Asn Val Trp Ile Cys Gln Arg 405 410 415 gct gct
cgg atc att ggc ctc gat att gct ggt att gac gtt gtc agc 1296 Ala
Ala Arg Ile Ile Gly Leu Asp Ile Ala Gly Ile Asp Val Val Ser 420 425
430 ccc gat att agt cag ccc ctg tct aaa gtt ggc ggt gtg att gtc gag
1344 Pro Asp Ile Ser Gln Pro Leu Ser Lys Val Gly Gly Val Ile Val
Glu 435 440 445 gtc aat gcc gct cct ggc ttt cgc atg cac acc aac ccc
agc caa ggg 1392 Val Asn Ala Ala Pro Gly Phe Arg Met His Thr Asn
Pro Ser Gln Gly 450 455 460 att gcc cgc aat gtt gcc gaa ccg gtg ttg
aat atg ctc ttt cca ccg 1440 Ile Ala Arg Asn Val Ala Glu Pro Val
Leu Asn Met Leu Phe Pro Pro 465 470 475 480 gga aca cct tgc cgc atc
ccg atc ttt gcc att acg ggg acc aat ggc 1488 Gly Thr Pro Cys Arg
Ile Pro Ile Phe Ala Ile Thr Gly Thr Asn Gly 485 490 495 aaa acc acc
acc acc cgt ctc att gcc cat atc tgc aaa caa acg ggg 1536 Lys Thr
Thr Thr Thr Arg Leu Ile Ala His Ile Cys Lys Gln Thr Gly 500 505 510
caa acc gtt ggc tac acc acc aca gac ggc atc tat att ggc gat tat
1584 Gln Thr Val Gly Tyr Thr Thr Thr Asp Gly Ile Tyr Ile Gly Asp
Tyr 515 520 525 ctg gtg gaa aaa gga gac acc acc ggc ccc caa agt gcc
caa ctg atc 1632 Leu Val Glu Lys Gly Asp Thr Thr Gly Pro Gln Ser
Ala Gln Leu Ile 530 535 540 ctg cag gac ccc acc gtt gag atc gcc gtt
ctc gaa acg gcg cga ggt 1680 Leu Gln Asp Pro Thr Val Glu Ile Ala
Val Leu Glu Thr Ala Arg Gly 545 550 555 560 ggg att ctc cgc tcc ggc
ttg ggc ttt gac cat tgt gat gtc ggg gtg 1728 Gly Ile Leu Arg Ser
Gly Leu Gly Phe Asp His Cys Asp Val Gly Val 565 570 575 gtg ctc aat
gtg cag gct gat cac ctt ggc ctt ggc gat att gac acc 1776 Val Leu
Asn Val Gln Ala Asp His Leu Gly Leu Gly Asp Ile Asp Thr 580 585 590
gtt gag cag ttg gcg gac tta aag gca gtg gtg gtg gaa tct gct tgg
1824 Val Glu Gln Leu Ala Asp Leu Lys Ala Val Val Val Glu Ser Ala
Trp 595 600 605 cca aat ggc tac gct gtg ttg aat gcc gat gat ccc cta
gtg gcg gca 1872 Pro Asn Gly Tyr Ala Val Leu Asn Ala Asp Asp Pro
Leu Val Ala Ala 610 615 620 atg gca cgc caa gtc aaa gct caa gtg gcc
tat ttc tcg atg gat ccc 1920 Met Ala Arg Gln Val Lys Ala Gln Val
Ala Tyr Phe Ser Met Asp Pro 625 630 635 640 cac aat ccc atc att cgg
cag cac atc cag cag ggg gga ctc gcc gct 1968 His Asn Pro Ile Ile
Arg Gln His Ile Gln Gln Gly Gly Leu Ala Ala 645 650 655 gtt tat gaa
aat ggc tac ctc tca att ttg aaa ggg gac tgg aca ctg 2016 Val Tyr
Glu Asn Gly Tyr Leu Ser Ile Leu Lys Gly Asp Trp Thr Leu 660 665 670
cgc att gag cag gca gaa aat gtg ccc att acc ctt ggc gct cga gca
2064 Arg Ile Glu Gln Ala Glu Asn Val Pro Ile Thr Leu Gly Ala Arg
Ala 675 680 685 agc ttt atg att gcc aat gcc ctc gct gcc agt cta gcg
gcc ttt gcc 2112 Ser Phe Met Ile Ala Asn Ala Leu Ala Ala Ser Leu
Ala Ala Phe Ala 690 695 700 caa ggc atc agt att gag cat att cgc gcc
gcc ttg acc acc ttc cga 2160 Gln Gly Ile Ser Ile Glu His Ile Arg
Ala Ala Leu Thr Thr Phe Arg 705 710 715 720 acc tcg gtg gag caa acc
ccc ggt cgg atg aac ctc ttt gat ttg ggg 2208 Thr Ser Val Glu Gln
Thr Pro Gly Arg Met Asn Leu Phe Asp Leu Gly 725 730 735 caa ttt agt
gtc ttg gtg gac tat gcc cac aat cca gca ggg tat gaa 2256 Gln Phe
Ser Val Leu Val Asp Tyr Ala His Asn Pro Ala Gly Tyr Glu 740 745 750
gcc att ggt gaa ttt gtc cag aaa tgg cca ggg cag cgc att ggt gtc
2304 Ala Ile Gly Glu Phe Val Gln Lys Trp Pro Gly Gln Arg Ile Gly
Val 755 760 765 gtt ggc gga cca ggc gat cgc cgc gat caa gac ttg gag
caa ctg ggg 2352 Val Gly Gly Pro Gly Asp Arg Arg Asp Gln Asp Leu
Glu Gln Leu Gly 770 775 780 gaa ctc tcg gcg aaa att ttt gat tgg atc
atc att aag gaa gat gat 2400 Glu Leu Ser Ala Lys Ile Phe Asp Trp
Ile Ile Ile Lys Glu Asp Asp 785 790 795 800 gat acc cgt ggc cgg cct
cgg ggc gat gcc gcc tat tgg att gag cgg 2448 Asp Thr Arg Gly Arg
Pro Arg Gly Asp Ala Ala Tyr Trp Ile Glu Arg 805 810 815 ggg gta cat
cac cac agt gtc cag cgg caa tac gac atc atc cat gac 2496 Gly Val
His His His Ser Val Gln Arg Gln Tyr Asp Ile Ile His Asp 820 825 830
gag gtg gca gcg att caa ttt gcc ctc gat cgc gct ccc aaa gga tcc
2544 Glu Val Ala Ala Ile Gln Phe Ala Leu Asp Arg Ala Pro Lys Gly
Ser 835 840 845 tta gtg gtg atc ttt cca gcg gaa gtc agc cgc acg att
caa ctg att 2592 Leu Val Val Ile Phe Pro Ala Glu Val Ser Arg Thr
Ile Gln Leu Ile 850 855 860 cgc cag cat cac caa cga ctc caa ggg gaa
acg atc aat ggc ttt cac 2640 Arg Gln His His Gln Arg Leu Gln Gly
Glu Thr Ile Asn Gly Phe His 865 870 875 880 agt gag gga agg ccc acc
agt ggt gat ctc aac ccc tcc atc ttt cat 2688 Ser Glu Gly Arg Pro
Thr Ser Gly Asp Leu Asn Pro Ser Ile Phe His 885 890 895 tag
2691
* * * * *